PROJECT ABSTRACT Inflammatory breast cancer (IBC), one of the most lethal forms of breast cancer, accounts for ~15% of all breast cancer deaths. IBC is particularly devastating in disadvantaged minority women who have both a higher incidence and poorer survival. Gene expression profiling of IBC tumors has identified the hedgehog(Hh)/GLI1 pathway as higher in patients with aggressive and relapsing IBC. We have previously demonstrated that GLI1 is highly expressed in IBC cells derived from an aggressive primary IBC tumor, and that down-regulation of GLI1 in these cells decreased cell proliferation, increased apoptosis and reduced migration. The objective of the proposed research is to identify those genes that are critical for GLI activation and function, including those that are both up- and downstream in of GLI1, to provide insight into the molecular mechanisms underlying or driving IBC. The hypothesis to be explored is that GLI1 and the genes that it regulates promote the growth and survival of IBC, and that identifying these genes may lead to the elucidation of novel targets. The long-term goal is to inform novel approaches for anti-tumor efficacy in IBC. This objective will be met by applying an innovative approach that includes utilization of high-throughput transcriptome sequencing (RNA-Seq), genetically engineered cell lines and human IBC tissue samples. This will become accomplished by the following Aims: 1) To identify gene expression differences between low and high GLI1 expressing IBC models including drug resistant and GLI1-antagonist treated IBC models. Differences in gene expression patterns will be determined by high-throughput transcriptome sequencing (RNA-Seq). 2) To confirm and validate those gene expression differences by qRT-PCR analysis including a comparison of IBC and non-IBC cell lines. To assess overlap of those differentially expressed genes with African American patient derived non-IBC cell lines. We will assess GLI1 expression and the expression of a subset of differentially expressed target genes identified in Aim 1 in archival IBC patient and control tissue samples accessed from the Duke Biospecimen Repository and Processing Core by immuno-histochemistry, and gene expression analysis. To define functional roles for these target genes, a subset will be knocked down by shRNA or knocked out using CRISPR/Cas9 and effects on IBC growth and survival will be determined. 3) Selective pharmacological inhibitors will be used to assess the role of previously identified pathways in activating GLI1 in IBC and non- IBC. We assess effects on IBC cell biology in a panel of functional and phenotypic assays. This proposed research will further the collaboration between Duke and NCCU on IBC and will support the development of an under-represented minority postdoctoral fellow performing research focused on transcriptional profiling of GLI1 activation pathways in IBC. Completion of these Aims will provide mechanistic insights into the consequences of GLI1 activation and antagonism in IBC, the downstream pathways involved and inform novel approaches for anti-tumor efficacy in IBC with the long term goal to translate new therapies for IBC into the clinic.